犬小孢子菌微量液基稀释法抗真菌药敏试验的方法学研究

目的对北京儿童医院的头癣患者分离所得的182株犬小孢子菌进行抗真菌药敏试验条件的探索。方法对致病真菌进行取样、培养、鉴定、分离和纯化,经微量稀释法测定8种常见抗真菌药物的最小抑菌浓度,并对原药敏方案进行适当调整。结果受试菌株的接种浓度为1~3×104CFU/mL。除伊曲康唑、联苯苄唑、阿莫罗芬外,使用沙氏液体培养基测定的药物最小抑菌浓度均较使用RPM I-1640液体培养基时药物浓度明显升高。孵育温度为28℃,孵育时间为5 d。结论各种药物的最小抑菌浓度(m in im al inh ib itory concentration,M IC)之间的差异有统计学意义,M38-A方案有较好的重复性和稳定性,可作为一种较好的实验室方法被应用。     

In vitro susceptibility testing of Aspergillus spp. against voriconazole, itraconazole, posaconazole, amphotericin B and caspofungin

Background During recent years, the incidence of serious infections caused by opportunistic fungi has increased dramatically due to alterations of the immune status of patients with hematological diseases, malignant tumors, transplantations and so forth. Unfortunately, the wide use of triazole antifungal agents to treat these infections has lead to the emergence of Aspergillus spp. resistant to triazoles. The present study was to assess the in vitro activities of five antifungal agents （voriconazole, itraconazole, posaconazole, amphotericin B and caspofungin） against different kinds of Aspergillus spp. that are commonly encountered in the clinical setting. Methods The agar-based Etest MIC method was employed. One hundred and seven strains of Aspergillus spp. （5 species） were collected and prepared according to Etest Technique Manuel. Etest MICs were determined with RPMI agar containing 2% glucose and were read after incubation for 48 hours at 35℃. MIC50, MIC90 and MIC range were acquired by Whonet 5.4 software. Results The MIC90 of caspofungin against A. fumigatus, A. flavus and A. nidulans was 0.094 μg/ml whereas the MIC90 against A. niger was 0.19μg/ml. For these four species, the MIC90 of caspofungin was the lowest among the five antifungal agents. For A. terrus, the MIC90 of posaconazole was the lowest. For A. fumigatus and A. flavus, the MIC90 in order of increasing was caspofungin, posaconazole, voriconazole, itraconazole, and amphotericin B. The MIC of amphotericin B against A. terrus was higher than 32 μg/ml in all 7 strains tested. Conclusions The in vitro antifungal susceptibility test shows the new drug caspofungin, which is a kind of echinocandins, has good activity against the five species of Aspergillus spp. and all the triazoles tested have better in vitro activity than traditional amphotericin B.     

过氧化氢对5种唑类药物体外抗耐药白假丝酵母菌的协同作用

目的考察过氧化氢(H2O2)对5种唑类药物:氟康唑(FLC),伊曲康唑(ICZ),酮康唑(KCZ),咪康唑(MCZ)和硫康唑(SCZ)体外抗耐药白假丝酵母菌作用的影响。方法利用8株对氟康唑耐药,2株对氟康唑敏感的白假丝酵母菌,采用微量液基稀释法测定药物单用时的最低抑菌浓度MIC80值,采用棋盘式微量液基稀释法测定两药联用时的MIC80值,在此基础上计算部分抑菌浓度指数(FICI)即协同指数,依据FICI值判断H2O2与唑类药物联用是具有协同、无关还是拮抗作用。结果对所有耐药菌株,两药联用的FICI值均小于0.5,H2O2与唑类药物联用显现出协同作用;对2株敏感菌株,两药联用的FICI值大于0.5、小于2,显示为无关作用。结论 H2O2和唑类药物(氟康唑,伊曲康唑,酮康唑,咪康唑,硫康唑)联用有协同抗耐药白假丝酵母菌作用。     

Antimicrobial activity of linezolid combined with minocycline against vancomycin-resistant Enterococci

Background Vancomycin-resistant Enterococci (VRE) cause serious infections that are difficult to treat.We carried out this study to determine the mutant prevention concentration (MPC) of linezolid when...     

多粘菌素B与亚胺培南联用对泛耐药鲍曼不动杆菌体外抗菌活性的研究

目的评价多粘菌素B与亚胺培南体外联合应用,对临床分离的30株泛耐药鲍曼不动杆菌的抗菌效应。方法采用微量肉汤稀释法、棋盘设计法测定多粘菌素B与亚胺培南单用及联合应用对临床分离的30株泛耐药鲍曼不动杆菌的最小抑菌浓度（MIC）,并计算FIC指数,判定联合效应：FIC≤0.5为协同作用,0.52.0为拮抗作用。结果多粘菌素B与亚胺培南联用后,药物对泛耐药鲍曼不动杆菌的MIC值均显著降低,FIC指数在0-0.5、0.5-1的百分率为：50%、40%。结论多粘菌素B与亚胺培南联用,对临床分离的30株泛耐药鲍曼不动杆菌抗菌作用以协同和相加为主,无拮抗作用。     

Antifungal activity of ibuprofen against aspergillus species and its interaction with common antifungal drugs

Background The incidence of invasive aspergillosis （IA） has increased in frequency in immunocompromised patients with a variety of diseases. The poor prognosis might be due to limited treatment option. This study aimed to evaluate antifungal activity of ibuprofen against clinical isolates of aspergillus species, as well as its interaction with azoles or with amphotericin B or with micafungin. Methods Antifungal activity of ibuprofen against 10 strains of Aspergillus fumigatus, Aspergillus flavus, and Aspergillus terreus were tested with both disk diffusion assay and standard broth microdilution method. To determine whether ibuprofen combined with itraconazole, voriconazole, amphotericin B, or micafungin had interactive effects on aspergillus spp., we used both disk diffusion assay and Chequerboard method. Results As for disk diffusion method, ibuprofen produced a zone of growth inhibition with diameters of （20.1±3.9） mm at 48 hours of incubation. As for broth microdilution method, the minimal inhibitory concentration （MIC） ranges of ibuprofen against aspergillus spp. were 1000-2000 μg/ml, and the minimal fungicidal concentration （MFC） ranges of that was 2000-8000 μg/ml. For 2 of 5 isolates, when ibuprofen combined with itraconazole or voriconazole, the zones of growth inhibition were larger than those of the individual drug. The results of Chequerboard method showed that fractional inhibitory concentration index （FICI） ranges were 1.125-2.500. Conclusions Ibuprofen is active against aspergillus spp.. And ibuprofen does not affect the in vitro activity of itraconazole, voriconazole, amphotericin B or micafungin against aspergillus spp.     

联苯苄唑的药效学研究实验室抗真菌活性测定

联苯苄唑是一种新的咪唑类衍生物,具有广谱抗真菌作用。本实验采用100株临床常见的真菌作了MIC测定。这些菌株对联苯苄唑普遍敏感,79%的菌株MIC≤4μg/ml。MIC值的大小受接种菌量的影响,接种量越大,MIC值越大。通过实验对比,联苯苄唑与咪康唑的抗真菌活性相近     

广东首例肾移植术后甄氏外瓶霉致皮肤及皮下组织暗色丝孢霉病

报道广东首例肾移植术后甄氏外瓶霉致皮肤及皮下组织暗色丝孢霉病临床、实验研究和治疗结果。患者男,66岁,右中指、腕部、前臂皮肤结节、脓疱5月余,无痒痛。脓液涂片显微镜下见分隔、分支,淡棕色菌丝;组织病理见炎性肉芽肿囊性变,其内散在真菌菌丝,PAS、六胺银染色亦见真菌菌丝;沙堡培养基26℃培养菌落呈榄黑色绒毛样,玻片小培养符合甄氏外瓶霉;对所分离菌株进行ITS序列分析,与甄氏外瓶霉同源性为100%;采用Etest法检测了6种抗真菌药物的MIC值,其中二性霉素B、伏立康唑、伊曲康唑、氟康唑敏感,5-氟胞嘧啶、卡泊芬净耐药。局部注射二性霉素B与口服伏立康唑联合抗真菌治疗,及时切除好转较慢及新发皮疹,治疗效果满意。本病例为广东省内首次报道甄氏外瓶霉所致暗色丝孢霉病,亦为肾移植术后所致该病的国内首次报道。     

二性霉素B及其脂质体在治疗侵袭性真菌感染中的地位和意义

目的:观察静脉用二性霉素B(AmB)及其脂质体(L-AmB)对侵袭性真菌感染(IF I)的临床疗效及安全性。方法:46例IF I的原发基础病包括血液病32例及非血液系统疾病14例。普通AmB用量为0.5~1.0 m g/(kg.d),L-AmB用量为0.5~2.0 m g/(kg.d),总剂量为215~2950m g,中位数455m g,疗程10~119d,中位数17d。并对进口AmB、L-AmB及国产AmB的抗真菌疗效和副作用进行了比较分析。结果:二性霉素B临床总有效率为73.9%,真菌清除率为71.0%。进口AmB、L-AmB及国产AmB的治疗有效率分别为76.5%、71.5%及73.3%。不良反应发生率中寒颤、发热19.6%,低血钾21.7%,肝、肾功能损害各为10.9%和15.2%。结论:早期诊断、早期治疗及经验性治疗是提高IF I患者生存率、降低死亡风险的关键。只要预防得当,合理用药,定期监测肝肾功能,二性霉素B仍是一相对较为安全有效的药物。     

胡桃楸提取物抗深部感染真菌的活性研究

目的探讨胡桃楸提物对常见深部感染真菌生长的抑制作用。方法以NCCLS方案检测胡桃楸提取物和伊曲康唑(ICZ)、氟康唑(FCZ)、两性霉素B(AMB)和5-氟胞嘧啶(5-FC)对白念珠菌、新生隐球菌和烟曲霉菌体外最小抑菌浓度(MIC)和最小杀菌浓度(MFC)。结果胡桃楸提取物对深部致病性真菌有较强的抑菌和杀菌作用,其作用强度与ICZ、FCZ相当,但逊色于AMB和5-FC。结论胡桃楸提取物对深部致病性真菌体外生长有较强的抑菌和杀菌作用。     

多肽APS体外抗病原真菌活性及其抗菌机理初探

目的 探讨抗菌肽APS的体外抗病原真菌的活性及可能的抗菌机理。方法 微量稀释法测定APS对不同真菌的最低抑菌浓度（MIC）及最低杀菌浓度（MBC），并检测APS对脂质平面膜电学性质的影响。结果 除过烟曲霉菌外，APS对其它受试病原真菌有较低的MIC及MBC，同时，经APS作用后，脂质平面膜上形成跨膜电流。结论 APS对多种常见病原真菌均有较强的抑制和杀灭作用。APS抗真菌的可能机制是在膜上持续形成孔道（pores)，破坏了膜的完整性，引起细胞内容物的外流，最终导致真菌的死亡。     

用鸡胚皮肤作绿脓杆菌粘附的定量研究——1.实验模型的建立

本文介绍用鸡胚皮肤作绿脓杆菌(PA)粘附的体外模型的方法。无菌操作取9～10日龄鸡胚背部皮肤3块(每块约20mm~2)与细菌孵育后用扫描电子显微镜对固定面积的细菌进行直接计数。粘附最适条件:接种菌量为10~7～10~8CFU/mL,孵育时间为1小时。结果表明,PA 与皮肤能发生明显粘附,有些 slime 菌株比 nonslime 菌株的粘附作用高10倍以上。加热或甲醛杀死的细菌不粘附皮肤。PA 抗血清也能抑制细菌粘附。其他革兰氏阴性杆菌(大肠杆菌,肺炎杆菌和变形杆菌)对皮肤的粘附作用较弱。这一模型对于研究 PA 与损伤皮肤所发生的早期感染过程将是十分有用的。     

常用抗厌氧菌药物对牙龈卟啉单胞菌的体外抗菌活性研究

目的 研究口腔临床常用抗菌药物对牙周炎主要病原菌牙龈卟啉单胞菌（Pg)的抗菌活性。方法 用从成人牙周炎患者龈下菌斑中分离的、通过PCR鉴定的106株Pg临床分离株,采用琼脂稀释法测定了甲硝唑、克林霉素和替硝唑对三种不同浓度Pg菌液的最小抑菌浓度（MIC）。结果 上述药物对Pg的MIC范围依次是≤0.25-8mg/L,≤0.06-8mg/L和≤0.06-4mg/L,MIC90依次为8mg/L,8mg/L,4mg/L.10^5CPU/ml,10^6CFU/ml和10^8CPU/ml三种菌液浓度对实验结果无明显影响。结论 Pg对这三种药都非常敏感,敏感性大小依次为替硝唑、克林霉素和甲硝唑。     

10种中药水煎剂对念珠菌的体外抑菌作用

目的 观察苦参、土槿皮、黄柏、百部、黄连、虎杖、黄芩、大黄、藿香、五倍子及其混合液体外抑制念珠菌的效果.方法 参照1997年美国国家临床实验标准化委员会M27-A方案中的酵母菌微量稀释法进行药敏试验.结果 同一种中药水煎剂及不同的单味中药水煎剂对不同的念珠菌标准菌株的最低抑菌浓度(minimum inhibitory concentration,MIC)各不相同,中药混合液对5种标准菌株的MIC最低,均≤0.031 25 g/ml.结论 不同的中药对不同的念珠菌的抑制作用各不相同,复方对念珠菌的抑制作用优于单味中药.     

10种中药水煎剂对念珠菌的体外抑菌作用

目的观察苦参、土槿皮、黄柏、百部、黄连、虎杖、黄芩、大黄、藿香、五倍子及其混合液体外抑制念珠菌的效果。方法参照1997年美国国家临床实验标准化委员会M27-A方案中的酵母菌微量稀释法进行药敏试验。结果同一种中药水煎剂及不同的单味中药水煎剂对不同的念珠菌标准菌株的最低抑菌浓度(minimuminhibitory concentration,MIC)各不相同,中药混合液对5种标准菌株的MIC最低,均≤0.031 25g/ml。结论不同的中药对不同的念珠菌的抑制作用各不相同,复方对念珠菌的抑制作用优于单味中药。     

Comparison of a glucose consumption based method with the CLSI M38-A method for testing antifungal susceptibility of Trichophyton rubrum and Trichophyton mentagrophytes

Background The prevalence of dermatophytoses and the development of new antifungal agents has focused interest on susceptibility tests of dermatophytes. The method used universally for susceptibility tests of dermatophytes was published as document （M38-A） in 2002 by the Clinical and Laboratory Standards Institute （CLSI）, dealing with the standardization of susceptibility tests in filamentous fungi, though not including dermatophytes especially. However, it is not a very practical method for the clinical laboratory in routine susceptibility testing. In this test, we developed a novel rapid susceptibility assay --glucose consumption method （GCM） for dermatophytes. Methods In this study, we investigated the antifungal susceptibilities of dermatophytes to itraconazole （ITC）, voriconazole （VOC）, econazole nitrate （ECN） and terbinafine （TBF） by glucose consumption method （GCM）, in comparison to the Clinical and Laboratory Standards Institute （CLSI） M38-A method. Twenty-eight dermatophyte isolates, including Trichophyton rubrum （T. rubrum） （n=-14） and Trichophyton mentagrophytes （T. mentagrophytes） （n=-14）, were tested. In the GCM, the minimum inhibitory concentrations （MICs） were determined spectrophotometrically at 490 nm after addition of enzyme substrate color mix. For the CLSI method, the MICs were determined visually. Results Comparison revealed best agreement for TBF against T. mentagrophytes and T. rubrum, since MIC range, MIC50, and MIC90 were identical from two methods. However, for ITC and VOC, GCM showed wider MIC ranges and higher MICs than CLSI methods in most isolates. For ECN against T. rubrum, high MICs were tested by GCM （0.125-16 pg/ml） but not M38-A method （0.5-1 IJg/ml）. The overall agreements for all isolates between the two methods within one dilution and two dilutions for ITC, VOC, ECN and TBF was 53.6% and 75.0%, 57.1% and 75.0%, 82.1% and 89.3%, and 85.7 and 85.7%, respectively. Conclusion Measurement of glucose uptake can predict the susceptibility of T. rubrum and T. mentagrophytes to ECN and TBF.     

N-乙酰半胱氨酸与常用抗菌药物对铜绿假单胞菌的联合抑菌作用

目的 研究N-乙酰半胱氨酸(NAC)与哌拉西林、环丙沙星、阿米卡星对铜绿假单胞菌的协同抑菌作用.方法 应用微量肉汤稀释法测定最低抑菌浓度(MIC);采用棋盘法检测NAC与哌拉西林、环丙沙星、阿米卡星对铜绿假单胞菌的联合抑菌作用,计算最小部分抑菌浓度指数.结果 NAC对大多数铜绿假单胞菌MIC在10-40mg/ml.对33.3%的铜绿假单胞菌,NAC与哌拉西林联合应用存在协同抑菌作用;对50%的铜绿假单胞菌,NAC与环丙沙星联合应用存在协同抑菌作用;NAC与阿米卡星联合应用对铜绿假单胞菌存在无关(86.7%)或拮抗作用(13.3%).结论 NAC对铜绿假单胞菌有抑菌作用,并与哌拉西林、环丙沙星存在一定协同抑菌作用.     

两性霉素B脂质体与两性霉素B抗念珠菌的MIC相关性探讨

目的 探讨两性霉素B脂质体(Ambisome)与两性霉素B(AmB)抗念珠菌的最低抑菌浓度(MIC)差别及其相关性.方法 参照美国国家临床实验室标准化委员会(NCCLS)M27-A推荐的常量稀释法,体外检测20株念珠菌临床分离株和1株标准菌株对AmB和Ambisome的MIC,对两药的MIC结果进行比较和相关性分析.结果 Ambisome抗念珠菌MIC与AmB相比在 1、 2个稀释度的符合率分别为52.4%(11/21)、23.8%(5/21),多数Ambisome的MIC值要高于AmB,平均增加0.82个稀释度;SPSS10.0统计软件等级相关分析显示两药MIC值有相关性(r=0.643,P＜0.01).结论 Ambisome体外抗念珠菌与AmB有相关性,为进一步根据AmB折点推测和建议Ambisome的折点提供依据.     

国产氨舒钠体外抗菌活性

目的 :比较国产氨舒钠 (ASN)和进口优立新 (ULX)制剂 (均由氨苄西林 (AMP)与舒马坦 (SBT)以 2 :1比例组成 )的体外抗菌活性。方法 :采用平皿二倍稀释法 ,多点接种仪点种细菌测定最小抑菌浓度 (MIC) ,并观察药物的MBC及接种菌量、pH、血清浓度改变对药物抗菌作用的影响。结果 :AMP与ASN或ULX对革兰阳性菌的MIC50 之平均比值分别为 4 8和 5 1;对革兰阴性菌的平均MIC50 比值分别为 6 3和 6 6 ;ASN和AMP的MBC为 2～ 4×MIC。接种菌量在 10 3 ～ 10 9CFU/ml、pH值在 5～ 9及血清浓度在 0 %～ 2 0 %的范围内 ,药物的MIC仅提高 1～ 4倍。结论 :SBT能增强氨苄西林的抗菌活性 ;接种菌量、pH、血清浓度改变对药物抗菌活性无明显影响。     

Clinical and laboratory evaluation of penicillin resistant Streptococcus pneumoniae in relation to the mutations of pbp 1a, pbp2b and pbp2x

A total of 210 isolates of Streptococcus pneumoniae (S. pneumoniae) were selected randomly to examine drug susceptibility which were obtained from out- and in-patients who visited Kurume University Hospital and other affiliated hospitals through May 1998 to September 1999. The prevalence of penicillin resistant S. pneumoniae in this study was 39.5% and was compatible with those of previous reports in Japan. From the clinical aspects, the resistant strains of S. pneumoniae have been shown not to be so highly virulent comparing with sensitive strains that only few severe or mortal cases had been reported. Carbapenems, glycopeptides, and fluoroquinolones were shown to be highly active against penicillin-resistant S. pneumoniae strains as evidenced by the low minimum inhibitory concentrations (MICs) though LVFX showed 4 micrograms/ml or higher MICs against some strains. Regarding to the mechanisms of penicillin resistance, penicillin binding proteins coding gene (pbp1a, pbp2x and pbp2b) mutations that cause modifications in these proteins were also examined for all isolates. The multivariate analysis showed statistically significant correlation between higher MIC of penicillin and cephem and pbp1a mutation while no significant contribution of pbp2x and pbp2b to the resistance was demonstrated. Additionally, no significant correlation between pbp mutation and MIC of carbapenem was observed. Furthermore, there were two highly penicillin resistant S. pneumoniae strains with no pbp mutations. Thus the pbp mutations alone were not responsible for the elevation of MIC all beta-lactams. Nevertheless the pbp mutations were detected in advance of actual MIC elevations by inducement experiment in vitro. It indicated that penicillin resistance might be detected earlier than conventional methods. In previous reports some other responsible genes for penicillin resistance were demonstrated. Therefore, it might be possible to presume exact values of MICs of beta-lactams against resistant strains of S. pneumoniae by detecting still unknown genes other than pbps.